1. Field of the Invention
The present invention relates to a method for diagnosing or predicting atherosclerosis based on a variation of the expression of an atherosclerosis biomolecular marker, a method for evaluating preventive or therapeutic effects of a compound on atherosclerosis, and a kit and an apparatus for carrying out such methods.
2. Background Art
The term “arteriosclerosis” collectively refers to diseases characterized by artery wall thickening caused by a variety of factors such as aging and lifestyle habits, decreased artery elasticity, and luminal stenosis. Cardiovascular diseases, such as myocardial infarction, and cerebrovascular diseases, such as cerebral infarction and cerebral hemorrhage, are regarded as arteriosclerosis-related diseases. In particular, atherosclerosis lesions (atheroma, plaque) comprises stable lesions rich in calcified fibrous tissue and unstable lesions rich in lipid and inflammatory cells having a relatively high risk of rupture. It is shown that most acute clinical findings regarding myocardial infarction, cerebral infarction, and the like are resulted from unstable atherosclerosis lesions. Therefore, safe and simple diagnosis and prevention of atherosclerosis, and establishment of effective therapeutic methods therefor are urgent needs (Koenig W. and Khuseyinova N., Arterioscler. Thromb. Vasc. Boil., 27:15-26, 2007).
At present, arteriosclerosis is diagnosed by invasive methods including angiography, and intravascular ultrasound, or noninvasive methods including ultrasound (particularly for the carotid artery and the femoral artery). It is difficult to distinguish stable and unstable atherosclerosis lesions by angiography, while on the other hand, it is relatively easy to distinguish them by angioscopy. However, both techniques are highly invasive. Therefore, less-invasive methods including the use of PET (positron emission tomography) and CTA (computerized tomography angiography) are being discussed. In any case, the use of an expensive and specified determination apparatuses and facilities is unavoidable, and the above techniques are available only in a limited number of institutions. In addition, such techniques can not easily process a plurality of specimens in parallel and lacks general versatility. Meanwhile, a clinical in vitro diagnostic technique for quantification of biomolecular markers whose levels significantly vary in blood or urine in the cases of certain diseases is a less invasive and highly versatile technique. It allows parallel processing of a plurality of specimens with the use of reagent kits and this can be carried out at a relatively low cost. This method does not require a specific measurement apparatus or facility and thus can be used in a general medical care facility. Also for atherosclerosis diagnosis, the development of a clinical in vitro diagnostic technique with the use of molecular markers has been expected.
For clinical in vitro diagnosis of atherosclerosis, a combined evaluation method using a risk factor group including obesity, hypertension, and lifestyle habits, in addition to main factors such as lipid markers (high levels of serum total cholesterol and LDL cholesterol and a low level of HDL cholesterol) has been suggested. Also, the Japan Arteriosclerosis Society describes the method in “Guidelines for Treatment of Hyperlipidemia (Kohshikessyo Chiryo Guideline).” The method has been used for risk evaluation and patient management in clinical practice. In addition, the relationship between variations in lipid markers and ischemic stroke has also been revealed. It is suggested that lipid markers can be used as indicators for the development of systemic arteriosclerosis in coronary arteries, cerebral arteries, and peripheral arteries and for the progression of lesions. However, there have been reports of a plurality of cases of patients with histories of myocardial infarction or with 50% coronary arterial stenosis discovered by a diagnostic method such as coronary angiography among patients whose laboratory values of lipid markers fell within the normal range and who were not found to have any specific risk factors. Therefore, the use of novel markers such as proteins and peptide molecules, in addition to lipid markers, has been discussed. For instance, CRP (C-reactive protein) is a protein showing a significant variation in a case involving an acute coronary artery disease. The blood CRP level increases up to as high as 300 μg/ml after 6 to 8 hours of the onset of the disease. Therefore, the applied use of CRP as a diagnosis marker in clinical practice for an ischemic heart disease or an unstable atherosclerosis lesion has been discussed (International Publication WO 98/43630; JP Patent Publication No. 2001-525058 A). However, it has been reported based on mass clinical analysis in Europe that evaluation with CRP alone is difficult due to its low specificity. Therefore, it is an important object to search for more specific protein/peptide molecular diagnosis markers with high levels of disease specificity and to use such markers in clinical practice.
CD166 (activated leukocyte cell adhesion molecule; ALCAM) is an immunoglobulin superfamily protein, which is a single-transmembrane protein belonging to the scavenger receptor cysteine rich superfamily. CD116 was identified as a CD6 ligand molecule. CD116 is known to be widely expressed in tissues and cells such as thymic epithelial cells, leukocytes, mesenchymal stem cells, and liver, pancreas, and brain tissues, and it regulates the intercellular signal transduction mechanism. It is thought that CD116 is widely present in humans, rodents, birds, zebrafish, and the like, regardless of species, and functions therein in a similar manner (Bowen M. A. et al., Eur. J. Immunol., 27:1469-1478, 1997). CD116 is also known to be expressed in cancer cells or tissues including prostate cancer, colorectal cancer, and melanoma, as well as other tissues, and to be involved in cancer metastasis. However, there are still no reports suggesting the involvement of CD116 in arteriosclerosis or unstable atherosclerosis lesions. Meanwhile, CD5L (apoptosis inhibitor expressed by macrophage; AIM) is a protein belonging to the scavenger receptor cysteine rich superfamily. CD5L was identified as a macrophage apoptosis inhibitory factor. Thereafter, it has been found that the arteriosclerosis lesion area significantly decreases in LDLR/CD5L double-deficient mice, which are arteriosclerosis-induced models, compared with LDLR-deficient mice, suggesting the involvement of CD5L in arteriosclerosis (Arai S. et al., Cell Metabolism, 1:201-213, 2005).